In article <guetlich.1129325084A at cony.gsf.de>, guetlich at haema103.gsf.de
(Markus Guetlich) wrote:
> We tried to transfect the monocytic cell lines U937 and THP-1 by
> electroporation with the electroporator bio rad gene pulser. The U937 could
> not be transfected by any variation of capacity and voltage. Perhaps anyone
> has more succeed in this, it would be nice to tell me. The transfection of
> THP-1 worked bad under our standard conditions for transfecting the raji
> B-cells (250 V,960uF). For any advice I thank you previously.
>> With best wishes
>> Klaus Witter
We transfect U937's routinely. We use 5X10+6 cells/500 ul with our DNA in
50 of RPMI 1640 (or alpha mem) with no additives. Mix and preinc for 5
min.
Electorporate in the 0.4 cm Biorad cuvettes in the Biorad GenePulser
witha Capacitance Extender set at 300 V, 960 uF, T=18-20 ms. Set on ice
15 min to allow the cells to recover. then grow your cells.
Since we use luciferase, the optimal time for incubations is 14 to 18
hours. This works for us.
SRL
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Stephen R. Lasky, Ph.D. Brown University/Roger Williams Medical Center
e-mail: Stephen_Lasky at brown.edu LandLine: 401-456-6572
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A man said to the Universe: "Sir, I exist!"
"However," replied the Universe, "the fact has not created in me a sense of obligation."
-- Stephen Crane
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